Gpr17 modulators,method of screening and uses thereof
The invention provides GPR17 modulators, methods of screening and use thereof for diagnosis and therapy of diseases or dysfunctions involving GPR17 activation, particularly ischemic brain damage.
The present invention relates to GPR17-modulating agents, in particular agents able to modify or block GPR17-receptor activity, and their use in the diagnosis and therapy of diseases or dysfunctions involving the same receptor.
BACKGROUND OF THE INVENTIONExtracellular nucleotides are universal and phylogenetically-ancient signalling molecules acting through specific membrane receptors: the seven ligand-gated P2X channels, and the eight G-protein-coupled P2Y receptors (the P2Y1,2,4,6,11,12,13,14 receptor subtypes)1,2. Endogenous ligands for P2Y receptors include adenine (ATP, ADP), uracil nucleotides (UTP, UDP) and, as more recently recognized, sugar nucleotides (e.g., UDP-glucose and UDP-galactose). Conversely, cysteinyl-leukotrienes (cysLTs) are peptide-conjugated lipid mediators generated by 5-lipoxygenase metabolism of arachidonic acid with established roles in bronchial asthma, acting through the CysLT1 and CysLT2 receptors3. Both P2Y receptors and CysLT receptors belong to the ∂ group of the GPCR rhodopsin family (the purin receptor cluster), which also includes the thrombin receptors and a large number of orphan GPCRs4. Among receptors in the purin cluster, GPR174 is one of the closest receptors to both P2Y and CysLT receptors, with a mean amino acid sequence identity of 31% with the eight recognized P2Y receptors and of 32 and 35% with CysLT1 and CysLT24.
STATE OF THE ARTThe identification of the nucleotide and amino acid sequences of cysteinyl-leukotriene GPR17 human receptor is reported in GB2360586. Also proposed therein are screening methods that may be used to identify agonists or antagonist modulating cysLT-receptor activity. According to GB2360586, the agents modulating cysLT-receptor activity may be used in the treatment and/or prophylaxis of several disorders.
DESCRIPTION OF THE INVENTIONThe invention is based on the finding that GPR17 represents a dualistic receptor responsive to unrelated families of signaling molecules acting through specific G-protein-coupled receptors, namely nucleotides and cysLTs. It has also been found that inhibition of GPR17 by either antagonist ligands or in vivo antisense technology in an animal ischemia model markedly reduces brain damages, indicating that GPR17 represents a common molecular target mediating the neuroinflammatory effects of nucleotides and cysLTs.
The possibility of modulating brain damage by interfering with a receptor responding to two distinct classes of ligands may significantly improve the therapeutic approach to diseases involving an excessive receptor activation, especially cardiovascular, neurodegenerative disorders and kidney ischemia. New chemical entities able to act on both the cys-LT and nucleotide component of GPR17, in particular, may prove extremely more effective in preventing brain damage and thus open up entirely new therapeutic strategies.
In a first embodiment, the invention provides a method for the identification of GPR17 modulators other than the leukotrienes or analogues thereof, which essentially comprises the following steps:
-
- 1) in vitro contacting GPR17 with a candidate compound, which is preferably a nucleotide derivative or analogue unable to interact with cysLT receptors;
- 2) determining the receptor response.
As used herein, “GPR17” indifferently indicates the human or rat receptor. The compounds able to bind the receptor and modulate its activity may be further investigated for their therapeutic potential.
The screening method may be applied to the identification of agonists, antagonists, inverse- or partial-agonists. In a preferred embodiment, the screening method is applied to the identification of compounds having receptor-antagonistic activity. In this case, step 1 above is carried out in the presence of a reference compound able to activate the receptor by binding to its nucleotide- or leukotriene-recognition site. Examples of compounds binding to the nucleotide recognition site of GPR17 include UDP, UDP-glucose and UDP-galactose. The assay can be carried out in a cell-based system or using cell preparations or fractions. Preferably, the pharmacological characterization of the receptor is carried out using the [35GTP]γS binding assay or the functional calcium imaging assay.
In the [35GTP]γS assay, upon agonist-binding the receptor undergo a conformational change which induces activation of the G-proteins responsible for signal-transduction. GPR17 activation can be assessed by testing the ability of exogenously added ligands to increase [35GTP]γS binding to purified membranes. In 1321N1 cells (which do not constitutively express any P2Y or CysLT receptors), heterologous hGPR17 expression induced the appearance of specific concentration-dependent responses to the cysLTs LTD4 and LTC4 and to the uracil nucleotides UDP, UDP-glucose and UDP-galactose. The ligand specificity of the human receptor was also confirmed in COS-7 and HEK-293 cells. Both AR-C69931MX (which has been reported as a selective P2Y12 and P2Y13 antagonist10,11,15), and the selective P2Y1-receptor antagonist MRS21797 concentration-dependently inhibited the [35S]GTPγS binding stimulated by UDP-glucose in membranes of cells expressing the human receptor. Conversely, the CysLT1 antagonists montelukast and pranlukast3,13 concentration-dependently inhibited the activation of human and rat receptors induced by LTD4. The different potencies between the two classes of ligands (nanomolar for cysteinyl leukotrienes and micromolar for nucleotides) suggests that GPR17 can undergo differential activation under specific physiological and pathological conditions. In particular, receptor activation by cysteinyl leukotrienes is believed to occur in physiological conditions, while in conditions of stress and injury the effect of nucleotides becomes important. In the latter situation, in fact, the concentration of nucleotides significantly increases in consequence of their release by hypoxic cells and their production by hydrolysis of nucleic acids in dead cells.
According to a further embodiment, the invention provides the use of GPR17-receptor antagonists for the preparation of a therapeutic agent for the treatment of diseases involving GPR17 activation, particularly neuroprotective, anti-inflammatory and preferably anti-ischemic agents for the treatment of cerebral, cardiac and renal ischemia. The antagonists may be identified with the method according to the invention, or they can be selected from the compounds having purinergic-receptor modulating activity. A comprehensive review of these latter can be found in Jacobson K. et al., “Molecular recognition at purine and pyrimidine nucleotide (P2) receptors”, Current Topics in Medicinal Chemistry 2004, vol. 4, pp. 671-686, herein entirely incorporated by reference. According to the invention, the antagonists MRS2179 (N6-methyl-adenosin-3′,5′-bis-phosphate, compound no. 46 in the reference) and AR-C69931-MX (N6-methylthio-ethyl-2-trifluoromethyl-ethylthio-adenosin-5′beta-methylene, γ-dichloromethylene trisphosphate, compound no. 57) are particularly preferred.
The invention further provides the use of combinations or associations of compounds acting on the GPR17-receptor sites respectively involved in the recognition of nucleotides and leukotrienes. The compounds acting on the GPR17-receptor site involved in the recognition of leukotrienes are preferably selected from:
-
- MK-571, 3-(3(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-(3-dimethylamino-3-oxo-propyl)thio)methyl)thio)propanoic acid, described in Jones T R et al., (1989) Pharmacology of L-660, 711 (MK-571): “a novel potent and selective leukotriene D4 receptor antagonist”. Can J Physiol Pharmacolo 67:17-28;
- Pranlukast ONO-1078, described in Obata T et al., (1985) New antagonists of leukotrienes: ONO-RS-411 and ONO-RS-347. Adv Prostaglandin Thromboxane Leukot Res 15:229-231;
- the inverse agonists MK-571 and Montelukast, described in Dupre D J et al., (2004) Inverse agonist activity of selected ligands of the cysteinyl-leukotriene receptor 1. J Pharmacol Exp Ther 309: 102-108.
The above bibliographic citations are herein entirely incorporated by reference.
For use in therapy, the GPR17 antagonists can be simultaneously administered, for example in a single pharmaceutical form or preparation, or separately, using different administration forms and routes. Besides the synthetic compounds (or “small molecules”) indicated above, the therapeutic approach to diseases involving GPR17 activation can be based on:
-
- expression vectors comprising the nucleotide sequence encoding the receptor protein, deletion or mutation variants thereof, for example plasmids, viruses or phages containing the regulatory sequences necessary for the correct expression of vector polynucleotide sequences (promoters, enhancers, initiation and termination sequences, polyadenylation sequences and, optionally, translation initiation and termination sequences);
- polypeptides having binding affinity to the receptor, able to modify the purinergic or leukotriene activity thereof, including synthetic oligopeptides, monoclonal or polyclonal antibodies recognizing and binding the GPR17 receptor;
- expression vectors comprising polynucleotides derived from the receptor-encoding sequence and governing the synthesis of antisense RNA;
- synthetic antisense polynucleotides as therapeutic agents. These polynucleotides may include molecules (aptamers) able to interact with the receptor or decoy molecules able to link nuclear proteins or regulatory sequences modulating the receptor expression on genomic DNA.
In an established animal model of permanent ischemic damage (the monolateral middle cerebral artery occlusion in the rat, MCAO), either montelukast or AR-C69931MX (GPR17 antagonists) markedly prevented increase of brain damage determined by Magnetic Resonance Imaging. The same result was observed when the expression of GPR17 was knocked down by utilizing antisense oligonucleotides. Of several antisense oligonucleotides designed on the sequence of rGPR17 mRNA, SEQ ID NO: 1 and SEQ ID NO: 2 (herein also referred to as oligo616 and oligo241, respectively) were able to reduce the in vitro expression of rGPR17 and, when intracerebroventricularly injected in rats, to significantly attenuate infarct size evolution in the lesioned cerebral site.
Therefore, in a particularly preferred embodiment, the invention provides antisense oligonucleotides according to SEQ ID NO: 1 and 2, and the use thereof for the preparation of a therapeutic agent for the treatment of ischemic brain damage.
The oligonucleotides sequences may be chemically modified or conjugated to improve their stability, in vivo delivery and pharmacokinetic profile. For example, the oligonucleotide backbone may be modified to contain 2′-O-(C1-C3) alkylribonucleotides, 2′-deoxyribonucleotides, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates, phosphinates, phosphoroamidates, thionophosphoroamidates, thionoalkylphosphonates or phosphotriesters groups. Other modifications may involve the sugar moiety, the internucleosidic bond or the purine or pyrimidine bases, e. g. by introducing purines and pyrimidines variously substituted on the heterocyclic rings, for example by alkyl, hydroxy- or halo-alkyl, halogen, hydroxyl, sulfur, amino or aza groups. Moreover, the oligonucleotides of the invention can be conjugated with different groups or functionalities able to increase their activity, distribution or cellular uptake. Such groups or functionalities include lipids, aliphatic chains, poliethilenglycol chains, polyamines and phospholipids.
For use in therapy, the pharmacological agents according to the invention, in particular small molecules, peptides and antisense oligonucleotides, may be suitably formulated together with physiologically acceptable excipients or carriers. Suitable pharmaceutical forms may vary depending on the specific compound or substance and on the administration route. The dosage of active ingredient will be determined on a case by case basis, depending on the severity of the disease to be treated and on the general conditions of the patients.
Suitable pharmaceutical compositions may be prepared following the indications provided in Remington's Pharmaceutical Sciences, XVIII Ed. Mack Publishing Co.
In a further embodiment, the invention relates to a diagnostic composition containing a compound, ligand, peptide, antibody or oligonucleotide able to interact with the purinergic site of the GPR17 receptor, particularly useful for the study of receptor functionality under physiological or pathological conditions.
The invention will be further illustrated by the following examples and by the annexed Figures.
1321N1 cells expressing hGPR17 were exposed in culture to either vehicle (empty columns) or 100 ng/ml PTX (black columns) for 18 hours before membrane preparation. [35S]GTPγS binding was then measured in the absence (basal) or presence of either nucleotides or cys-LTs, as indicated. Data are the mean of 3 experiments run in triplicate. In membranes preincubated with PTX, responses induced by agonists (black column) were in all cases significantly lower (P<0.001) with respect to responses detected with the same compounds in membranes from untreated cells (white columns).
Cell culture and treatments. Human astrocytoma cells (ADF cells), 1321N1, COS-7 and HEK-293 cells were cultured as previously described11,24. For [35S]GTPγS, 106 1321N1, COS-7 or HEK-293 cells were seeded on 75 cm2 flasks and transfected by the calcium phosphate precipitation method as previously described11. For experiments with anti-sense oligonucleotides (see also below), after plating HEK-293 cells were trasfected with the various oligos in Fugene™. For calcium imaging studies, 1321N1 and COS-7 cells were seeded on 2.4-cm diameter glass coverslips (100×103 cells). In selected experiments, cells were exposed to 100 ng/ml PTX (Sigma) for 18 h before membrane preparation, to inhibit PTX-sensitive Gi proteins. For treatment of cultured cells with oligonucleotides, 13×104 HEK-293 cells were seeded on 9 cm2 dishes and treated as described in Supplementary
Reagents. All culture media and sera were from Celbio. All reagents for RT-PCR, cloning and transfection were from Invitrogen, with the exception of Fugene™, which was from Roche Diagnostics. LTD4 was purchased from Cayman Chemical Co. (Ann Arbor, Mich.). Anti-sense oligonucleotides were selected according to the general criteria for oligo designing and synthetized by MWG-Biotech AG. In particular, thermodynamic criteria were set according to previous indications25 and care was taken to avoid internal loop, palindrome of 6 or more base pairs, nucleotides repetition (more then 3 base pairs), and where possible, a AGGG consensus sequence shown to target RNAse degradation was included in the designed oligos (see oligo 616 and 241 in Supplementary
Total RNA isolation and PCR Analysis. Total RNA was extracted using the TRIZOL® Reagent (Invitrogen) according to manufacturer's instructions. Retrotranscription to cDNA and PCR reactions were carried out as previously described11.
Cloning and heterologous expression of human and rat GPR17. The following specific oligonucleotide PCR-primers external to the open reading frame (ORF) of the previously reported human receptor sequence (GenBank accession no U33447) were utilized to amplify a 1087 bp product from human astrocytoma ADF cell:
The amplification product was cloned into a pcDNA3.1 expression vector using the pcDNA3.1/V5-His©TOPO® TA Expression Kit (Invitrogen, Milan, Italy). Interrogation of the rat HTGS database with the nucleotidic sequence of hGPR17 revealed the presence of an highly similar (89% identical) sequence in chromosome 18 supercontig (Genbank accession No.: AC112062). By utilizing specific oligonucleotide primers external to the putative 1020 bp ORF of the rat sequence we also cloned rGPR17 from rat brain. Constructs were verified by sequencing using the Applied Biosystems Terminator cycle sequencing kit. A partial sequence of the mouse ortholog of GPR17 is reported in Genbank (AY255543) and the complete sequence 98% identical to the rat receptor was found in a BAC clone (AC131761) using the rat sequence as a probe in the mouse HTGS database.
[35S]GTPγS Binding Assay. 1321N1 cells, COS-7 and HEK-293 cells (control and transfected cells) were homogenized in 5 mM TRIS-HCl, 2 mM EDTA, pH 7.4 and centrifuged at 48000 g for 15 min at 4° C. The resulting pellets (plasma membranes) were washed in 50 mM TRIS-HCl, 10 mM MgCl2, pH 7.4 and stored at −80° C. until used. Measurement of nucleotide-stimulated [35S]GTPγS binding to membranes of cells expressing the human or rat GPR17 receptor was performed as previously described9-11.
Functional calcium imaging assay. Measurements of intracellular calcium concentrations ([Ca2+]i) were carried out as previously described27. Forty-eight h after transfection, 1321N1 and COS-7 cells were loaded with 2 μM Fura-2 pentacetoxy methylester in Krebs-Ringer solution, washed and transferred to the recording chamber of an inverted microscope (Axiovert 100; Zeiss, N.Y.) equipped with a calcium imaging unit. Polychrome IV (TILL Photonics, Germany) was used as light source. Fura-2 and EGFP fluorescence images were collected with a PCO Super VGA SensiCam (Axon Instruments, Forest City, Calif.) and analyzed with the Axon Imaging Workbench 2.2 software (Axon Instruments). Images were acquired at 1-4 340/380 ratios/s.
Induction of focal brain ischemia in the rat. Male Sprague-Dawley rats (Charles River) underwent permanent middle cerebral artery occlusion (MCAo) as previously described28,29. Drug treatments were as follows: montelukast (2 mg/kg, intravenously, i.v., single bolus of 200 μl in physiological solution) and AR-C69931MX (4.5 μg/animal, intracerebroventricularly, i.c.v., 5 μl in physiological solution) were administered 10 minutes after MCAo. AR-C was utilized here to simply test the involvement of GPR17 in brain ischemia, and, being a very polar molecule which is likely to very poorly permeate the blood brain barrier, it was administered i.c.v. Oligo-616 and oligo-scramble (400 ng in 5 μl of physiological solution) were administered i.c.v three times to each rat 48, 24 h before and 10 minutes after MCAo. Control groups received corresponding vehicle i.c.v.
Magnetic Resonance Imaging analysis. MRI measurements were performed 2, 24 and 48 h after MCAo using a 4.7T, vertical superwidebore magnet of a Bruker AMX3 spectrometer with micro imaging accessory. Animal preparation, image acquisition, trace of the diffusion tensor map computation, ischemic volume determination and progression of the ischemic damage over time was as previously described30.
Statistical analysis. For [35S]GTPγS binding data, analysis and graphic presentation was performed by the non-linear multipurpose curve-fitting computer program Graph-Pad Prism (GraphPad). For calcium imaging, data were normalized to the mean F340/380 increase recorded in control cells. All data are presented as mean±SEM of 4-18 experiments run in triplicate. Statistical analysis was performed by either Student's t test or one-way ANOVA (Scheffe' test). Significance refers to results where P<0.05 was obtained.
Results
GPR17: a Close Relative of Both P2Y and CysLT Receptors
In search for the natural ligand of GPR17, we first cloned and analyzed the coding sequences from the human and rat receptors. The previously unidentified rat hortholog displayed a 89% aminoacid identity with the human sequence (
Functional Characterization Unveils the Dual Pharmacology of GPR17
To identify the endogenous ligand of GPR17, the cDNAs from human and rat GPR17 were cloned into the mammalian expression vector pcDNA3.1 and transfected in 1321N1, COS-7 and HEK-293 cells for functional characterization. GPR17 activation was assessed by testing the ability of exogenously-added ligands to increase [35S]GTPγS binding to purified membranes obtained from transfected cells9-11. In 1321N1 cells (which do not constitutively express any functional P2Y or CysLT receptors12 (Rovati G E & Abbracchio M P, unpublished observations), heterologous hGPR17 expression (
Table 1. Potency of various ligands on [35S]GTPγS binding to membranes obtaind from 1321N1 cells-transfected with human or rat GPR17. This table summarized the EC50 and IC50 values for agonists and antagonists respectively, of the various ligand tested in vitro on the recombinant human or rat GPR17, upon expression in 1321N1 cells. All data represent the mean of triplicate determinations from 4-9 independent experiments.
Inhibition of GPR17 Prevents Evolution of Ischemic Brain Damage
In order to characterize the pathophysiological roles of GPR17, based on data suggesting massive accumulation of both cys-LTs and nucleotides in traumatic and ischemic tissues (Burnstock & Knight, 2004; Ciceri et al., 2001, Ohtsuki et al., 1995) and also based on our previous results demonstrating restricted receptor expression in organs that typically undergo ischemic damage (
- 1. Burnstock, G. & Knight G. E. Cellular distribution and functions of P2 receptor subtypes in different systems. Rev. Cytol. 240, 31-304 (2004).
- 2. Abbracchio, M. P. et al. Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends Pharmacol. Sci. 24, 52-55 (2003).
- 3. Brink, C. et al. International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors. Pharmacol. Rev. 55, 195-227 (2003).
- 4. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. & Schioth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256-72 (2003).
- 5. Erb, L. et al. Site-directed mutagenesis of P2U purinoceptors. Positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity. J. Biol. Chem. 270, 4185-4188 (1995).
- 6. Jiang, Q. et al. A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor. Mol. Pharmacol. 52, 499-507 (1997).
- 7. Jacobson, K. A., Jarvis, M. F. & Williams M. Purine and pyrimidine (P2) receptors as drug targets. J. Med. Chem. 45, 4057-4093 (2002).
- 8. Blasius, R., Weber, R. G., Lichter, P. & Ogilvie, A. A novel orphan G protein-coupled receptor primarily expressed in the brain is localized on human chromosomal band 2q21. J. Neurochem. 70, 1357-1365 (1998).
- 9. Kotani, M. et al. Functional characterization of a human receptor for neuropeptide FF and related peptides. Br. J. Pharmacol. 133, 138-44 (2001).
- 10. Marteau, F. et al. Pharmacological characterization of the human P2Y13 receptor. Mol. Pharmacol. 64, 104-12 (2003).
- 11. Fumagalli, M. et al. Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y(13) receptor. Biochem. Pharmacol. 68, 113-124 (2004).
- 12. Communi, D. et al. Identification of a novel human ADP receptor coupled to G(i). J. Biol. Chem. 276, 41479-41485 (2001).
- 13. Capra, V. Molecular and functional aspects of human cysteinyl leukotriene receptors. Pharmacol. Res. 50, 1-11 (2004).
- 14. Herold, C. L., Li, Q., Schachter, J. B., Harden, T. K. & Nicholas, R. A. Lack of nucleotide-promoted second messenger signalling responses in 1321N1 cells expressing the proposed P2Y receptor, p2y7. Biochem. Biophys. Res. Commun. 235, 717-721 (1997).
- 15. Ingall, A. H. et al. Antagonists of the platelet P2T receptor: a novel approach to antithrombotic therapy. J. Med. Chem. 42, 213-220 (1999).
- 16. Ciceri, P., Rabuffetti, M., Monopoli, A. & Nicosia S. Production of leukotrienes in a model of focal cerebral ischaemia in the rat. Br. J. Pharmacol. 133, 1323-1329 (2001).
- 17. Ohtsuki, T. et al. Reperfusion induces 5-lipoxygenase translocation and leukotriene C4 production in ischemic brain. Am. J. Physiol. 268, H1249-H1257 (1995).
- 18. Stein, C. A. The experimental use of antisense oligonucleotides: a guide for the perplexed. J. Clin. Invest. 108, 641-644 (2001).
- 19. Tepper, J. M., Sun, B. C., Martin, L. P. & Creese I. Functional roles of dopamine D2 and D3 autoreceptors on nigrostriatal neurons analyzed by antisense knockdown in vivo. J. Neurosci. 17, 2519-2530 (1997).
- 20. Van Oekelen, D., Luyten, W. H. & Leysen, J. E. Ten years of antisense inhibition of brain G-protein-coupled receptor function. Brain Res. Brain Res. Rev. 42, 123-42 (2003).
- 21. Kenakin, T. P., Bond, R. A. & Bonner, T. I. Definition of pharmacological receptors. Pharmacol. Rev. 44, 351-362 (1992).
- 22. Kenakin, T. Principles: receptor theory in pharmacology. Trends Pharmacol. Sci. 25, 186-92 (2004).
- 23. Mellor, E. A., Maekawa, A., Austen, K. F. & Boyce, J. A. Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells. Proc. Natl. Acad. Sci. USA. 98, 7964-9 (2001).
- 24. Brambilla, R., Ceruti, S., Malorni, W., Cattabeni, F. & Abbracchio, M. P. A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes. J. Auton. Nerv. Syst. 81, 3-9 (2000).
- 25. Matveeva, O. V. et al. Thermodynamic criteria for high hit rate antisense oligonucleotide design. Nucleic Acids Res. 31: 4989-9 (2003).
- 26. Smith, L. et al. Rational selection of antisense oligonucleotide sequences. Eur J Pharm Sci. 11: 191-8. (2000).
- 27. Fumagalli, M. et al. Nucleotide-mediated calcium signalling in rat cortical astrocytes: Role of P2X and P2Y receptors. Glia 43, 218-03 (2003).
- 28. Tamura, A., Graham, D. I., McCulloch, J. & Teasdale, G. M. Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab. 1, 53-60 (1981).
- 29. Sironi, L. et al. Treatment with statins after induction of focal ischemia in rats reduces the extent of brain damage. Arterioscler. Thromb. Vasc. Biol. 23, 322-7 (2003).
- 30. Guerrini, U., et al. New insights into brain damage in stroke-prone rats: a nuclear magnetic imaging study. Stroke 33, 825-830 (2002).
Claims
1-17. (canceled)
18. A method for the identification of a GPR17 modulator, which essentially comprises the steps of:
- a) contacting GPR17 with a candidate compound, the latter being a nucleotide derivative or analog unable to interact with cysteinyl leukotriene receptors;
- b) determining the receptor response.
19. A method according to claim 18, which is carried out in a cell-based system or using cell preparations or fractions.
20. An method according to claim 18, wherein the receptor response is determined by means of the [35S]GTPS binding assay.
21. A method according to claim 18, for the identification of receptor antagonists.
22. A method according to claim 18, wherein step (a) is carried out in the presence of a compound able to bind to GPR17 nucleotide-recognition site.
23. A method according to claim 22, wherein said compound is selected from UDP, UDP-glucose and UDP-galactose.
24. An antisense oligonucleotide selected from SEQ ID NO: 1 and SED ID NO: 2, or a functional analog thereof.
25. A method for the treatment of disorders involving GPR17 activation, comprising administering an effective amount of a GPR17 antagonist that interacts with the receptor nucleotide binding site.
26. The method according to claim 24, wherein said GPR17 antagonist compound selected from N6-methyl-adenosin-3′,5′-bis-phosphate and N6-methylthio-ethyl-2-trifluoromethyl-ethylthio-adenosin-5′beta-methylen-dichloromethylene trisphosphate.
27. A method leukotriene receptor-binding site, for the preparation of a therapeutic agent for the treatment of disorders involving GPR17 activation.
28. The method according to claim 24, wherein the GPR17 antagonist is a compound selected from N6-methyl-adenosin-3′,5′-bis-phosphate and N6-methylthio-ethyl-2-trifluoromethyl-ethylthio-adenosin-5′beta-methylen-dichloromethylene trisphosphate in combination with a compound selected from Montelukast, Pranlukast and 3-(3(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-(3-dimethylamino-3-oxo-propyl)thio)methyl)thio)propanoic acid.
29. A diagnostic device comprising GPR17 nucleotide binding site.
30. A neuroprotective, anti-inflammatory and anti-ischemic agent, comprising a GPR17 antagonist.
31. A method for the treatment of cerebral, cardiac and renal ischemia, comprising administering an effective amount of a GPR17 antagonist.
32. The method according to claim 30 wherein the GPR17 antagonist is an antisense oligonucleotide selected from SEQ ID NO: 1 and SEQ ID NO: 2.
33. A method for the treatment of ischemic brain damage, comprising administering an effective amount of an antisense oligonucleotide selected from SEQ ID NO: 1 and SEQ ID NO: 2
34. A method for the treatment of ischemic brain, heart and renal damage, comprising administering an effective amount of small interference RNAs directed against SEQ ID NO: 1 and SEQ ID NO: 2
Type: Application
Filed: Oct 17, 2005
Publication Date: Jun 18, 2009
Patent Grant number: 8158593
Inventors: Maria Pia Abbracchio (Milano), Paolo Ciana (Gravellona Toce), Gianenrico Rovati (Pavia), Claudia Martini (Pisa), Maria Letizia Trincavelli (Pontedera), Claudia Verderio (Milano)
Application Number: 11/665,835
International Classification: A61K 31/7088 (20060101); C12Q 1/68 (20060101); C12M 1/34 (20060101); A61P 3/10 (20060101);